CAJ | 학술논문

目的:用基因工程手段获得有活性的抗独特型抗体I50,并在体外鉴定其活性.方法:以fuse5-I50为模板,用PCR方法扩增出抗独特型抗体I50基因,并将其插入到pET25b(+)中构建原核表达载体pET25b-I50.含有重组质粒pET25b-I50的菌株E.coli BL21(DE3)经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导能够得到有效表达.采用Western印迹鉴定I50蛋白的表达,采用透析复性方法恢复I50蛋白的活性,并用Dot-ELISA法,淋巴细胞增殖实验鉴定其活性.结果:成功的构建了原核表达载体pET25b-I50,并经IPTG诱导获得了I50 蛋白,该蛋白以包涵体形式高效表达,经纯化后纯度达90%以上.Western印迹鉴定表达的蛋白相对分子质量约15000,与预期相符.Dot-ELISA法鉴定复性后的蛋白已恢复活性,并能够刺激淋巴细胞的增殖,且呈剂量依赖关系.结论:成功获得了有活性的I50抗独特型抗体,为其应用于鼻咽癌的治疗鉴定了基础.
목적:용기인공정수단획득유활성적항독특형항체I50,병재체외감정기활성.방법:이fuse5-I50위모판,용PCR방법확증출항독특형항체I50기인,병장기삽입도pET25b(+)중구건원핵표체재체pET25b-I50.함유중조질립pET25b-I50적균주E.coli BL21(DE3)경이병기-β-D-류대필남반유당감(IPTG)유도능구득도유효표체.채용Western인적감정I50단백적표체,채용투석복성방법회복I50단백적활성,병용Dot-ELISA법,림파세포증식실험감정기활성.결과:성공적구건료원핵표체재체pET25b-I50,병경IPTG유도획득료I50 단백,해단백이포함체형식고효표체,경순화후순도체90%이상.Western인적감정표체적단백상대분자질량약15000,여예기상부.Dot-ELISA법감정복성후적단백이회복활성,병능구자격림파세포적증식,차정제량의뢰관계.결론:성공획득료유활성적I50항독특형항체,위기응용우비인암적치료감정료기출.
Objective To obtain I50 anti-idiotype antibody and identify its activity in vitro.Methods I50 anti-idiotype (Id) antibody gene was amplified from the template of fuse 5-I50 by PCR to construct a prokaryotic expression vector pET25b-I50. The expression of pET25b-I50 in E. coli BL21(DE3) was induced by isopropylthio-β-D-galactopyranoside (IPTG) and was confirmed by SDS-PAGE and Western blot with Ab1(FC2) monoclonal antibody and an anti-hexahistidine tag antibody. The method of dialysis refolding was used to restore the activity of I50 anti-Id antibody, which was measured by Dot-ELISA and lymphocyte proliferation assay. Results The recombinant vector was successfully constructed and the recombinant protein was successfully expressed and purified with 90% purity. The relative molecular weight of the expressed protein was 15 kD, which was in accordance with expectation. The activity of I50 anti-Id antibody could be restored and could promote the proliferation of lymphocyte in a dose-dependent manner. Conclusion These results suggested that I50 anti-Id protein vaccine is likely an option in the therapy against nasopharyngeal carcinoma in vivo.